1. Field of the Invention
This invention relates to proteins having asymmetric hydrolase activity for 4-substituted 1,4-dihydropyridine derivatives, genes encoding the proteins, and the use of the proteins.
2. Description of the Related Art
Of 4-substituted 1,4-dihydropyridine derivatives, those in which two different substituents are attached to the 3- and 5-positions of the dihydropyridine ring have an asymmetric carbon atom at the 4-position thereof and exist in the form of two optical isomers. As a result of investigations on the biological properties of these compounds, it has recently been reported that there is a difference in pharmacological activity, in vivo dynamic behavior, safety and other properties between the optical isomers of each compound [K. Tamazawa et al., J. Med. Chem., Vol. 29, 2504 (1986)]. Where such a compound having an asymmetric carbon atom is used as a drug, the conception of administering only one isomer favorable for use as a drug is being popularized so that no extra burden may be imposed on the living body. From this point of view, various processes for the preparation of optically active 3,5-disubstituted derivatives of 4-substituted 1,4-dihydropyridines are being investigated.
As a general method for the synthesis of optically active 4-substituted 1,4-dihydropyridine derivatives (in particular, 3,5-dicarboxylic acid monoesters), there is known a process comprising the steps of using a (4R)-1,4-dihydropyridine-3 or 5-carboxylic acid monoester as an intermediate and introducing a desired ester group thereinto [A. Ashimori et al., Chem. Pharm. Bull., Vol. 39, 108 (1991)]. Well-known methods for the preparation of such optically active intermediates [i.e., (4R)-1,4-dihydropyridine-3,5-carboxylic acid monoesters] include the chemical process of Shibanuma et al. [Chem. Pharm. Bull., Vol. 28, 2809 (1980)], as well as the enzymatic process of Achiwa et al. [Tetrahedron Letters, Vol. 32, 5805 (1991)] and the enzymatic process of Charles J. Sih et al. [Tetrahedron Letters, Vol. 32, 3465 (1991)].
However, the above-described processes do not always make it possible to prepare the desired compounds efficiently. Consequently, the present inventors have proposed a simpler and efficient process for converting a 4-substituted 1,4-dihydropyridine-3,5-dicarboxylic acid diester to the corresponding 3,5-dicarboxylic acid monoester efficiently by using a culture of a microorganism such as bacteria of the genera Streptomyces, Paecilomyces, Botryodioplodia and Alternaria (see the pamphlet of PCT International Publication No. 94/05637).
However, there still exists a need for the provision of a more efficient means for the preparation of optically active 4-substituted 1,4-dihydropyridine-3,5-dicarboxylic acid monoesters.
Accordingly, an object of the present invention is to provide isolated genes which are conducive to the efficient preparation of the aforesaid optically active compounds, expression plasmids containing such genes, transformants obtained by using such plasmids, and proteins obtained by culturing such transformants and having asymmetric hydrolase activity for 4-substituted 1,4-dihydropyridine derivatives, as well as means associated with the use thereof.
The present inventors have succeeded in cloning a DNA fragment containing a gene encoding a protein having asymmetric hydrolase activity for 4-substituted 1,4-dihydropyridine derivatives (hereinafter referred to as xe2x80x9c1,4-DHPDsxe2x80x9d), the chromosomal DNA of Strepto-myces viridosporus belonging to the aforesaid genus Streptomyces, and expressing the aforesaid gene.
Moreover, as a result of the sequence determination of the aforesaid gene and the characterization of the protein obtained by the expression thereof, there has been obtained extensive information on the relationship between the primary structure of the protein and the aforesaid asymmetric hydrolase activity thereof, and the like. Furthermore, as a result of retrieval on sequence homology and the like by using a database including the amino acid sequences of the aforesaid protein and various other proteins, it has also been found that the protein of the present invention has a certain degree of amino acid sequence homology with subtilisin [see Jacobs, M. et al., Nucleic Acids Res. (1985), 13:8913-8926] and is presumed to have Asp, His and Ser as active sites.
Thus, according to the present invention, there is provided an isolated gene containing at least that portion of the amino acid sequence represented by SEQ ID NO:7 which extends from Asp(29) to Ser(238), and encoding a protein having asymmetric hydrolase activity for 1,4-DHPDs, or a DNA fragment hybridizable with the gene.
Moreover, the present invention also provides a plasmid constructed by integrating the aforesaid gene into a vector, a transformant obtained by using this plasmid, and a process for the preparation of a protein having asymmetric hydrolase activity for 1,4-DHPDs by culturing the aforesaid transformant.
Furthermore, the present invention also provides a process for the preparation of optically active (4R)-1,4-dihydro-2,6-dimethyl-4-(nitrophenyl)pyridine-3,5-dicarboxylic acid monoester derivatives which comprises bringing a protein having such enzyme activity into contact with a compound of the formula 
wherein R is a lower alkanoyl group, a heterocyclic carbonyl group, a halogen-substituted acetyl group, an alkoxyacetyl group, an aryloxyacetyl group, a substituted or unsubstituted phenyl-lower alkanoyl group, a phenyl-substituted or unsubstituted lower alkenoyl group, an alkoxy- or alkenyloxycarbonyl group, an aralkyloxycarbonyl group or organic sulfonyl group, and n is an integer of 2 to 4, or a salt thereof in an aqueous medium; and recovering an optically active 4-substituted 1,4-dihydropyridine compound of the formula 
wherein R and n have the same meanings as described above, or a salt thereof.
Some of the aforesaid dicarboxylic acid monoester derivatives prepared in this manner are useful as intermediates for the synthesis of optically active 1,4-DHPDs which are well known per se and useful as prophylactic and therapeutic agents for ischemic heart diseases, hypertension and the like.